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Abstract

We present a novel concept adaptable to any kind of STED microscope in order to expand the limited number of compatible dyes for performing super resolution imaging. The approach is based on an intensity modulated excitation beam in combination with a frequency dependent detection in the form of a standard lock-in amplifier. This enables to unmix fluorescence signal originated by the excitation beam from the fluorescence caused by the STED beam. The benefit of this concept is demonstrated by imaging biological samples as well as fluorescent spheres, whose spectrum does not allow STED imaging in the conventional way. Our concept is suitable with CW or pulsed STED microscope and can thereby be seen as a general improvement adaptable to any existing setup.

Figures (5)

Simulation on the effect of residuals of fluorescence on STED microscopy performances. Confocal image of a group of subdiffracted beads (upper row). Middle row: STED image of a single point emitter (effective point spread function) by assuming kex = 0.01*ksted and k’sted = 0 (left), k’sted = 0.1*ksted (middle) and k’sted = 0.5*ksted (right). Bottom row: STED image of a group of point emitters in sub-diffraction distance imaged by the above described effective point spread functions. Scale bars: 100 nm.

(a) Excitation and Emission spectrum of red fluorescent spheres (Invitrogen). (b) fluorescence depletion curve measured in the classical mode (black) and in the modSTED configuration (red) for two different excitation powers of 7.1µW (squares) and 11.4µW (triangles). All power values are measured in front of the back aperture of the objective lens.